Genome-Wide Mycobacterium tuberculosis Variation (GMTV) Database: A New Tool for Integrating Sequence Variations and Epidemiology

Background Tuberculosis (TB) poses a worldwide threat due to advancing multidrug-resistant strains and deadly co-infections with Human immunodeficiency virus. Today large amounts of Mycobacterium tuberculosis whole genome sequencing data are being assessed broadly and yet there exists no comprehensive online resource that connects M. tuberculosis genome variants with geographic origin, with drug resistance or with clinical outcome. Description Here we describe a broadly inclusive unifying Genome-wide Mycobacterium tuberculosis Variation (GMTV) database, (http://mtb.dobzhanskycenter.org) that catalogues genome variations of M. tuberculosis strains collected across Russia. GMTV contains a broad spectrum of data derived from different sources and related to M. tuberculosis molecular biology, epidemiology, TB clinical outcome, year and place of isolation, drug resistance profiles and displays the variants across the genome using a dedicated genome browser. GMTV database, which includes 1084 genomes and over 69,000 SNP or Indel variants, can be queried about M. tuberculosis genome variation and putative associations with drug resistance, geographical origin, and clinical stages and outcomes. Conclusions Implementation of GMTV tracks the pattern of changes of M. tuberculosis strains in different geographical areas, facilitates disease gene discoveries associated with drug resistance or different clinical sequelae, and automates comparative genomic analyses among M. tuberculosis strains

A Comparison of the Sensititre MycoTB Plate, the Bactec MGIT 960, and a Microarray-Based Molecular Assay for the Detection of Drug Resistance in Clinical <i>Mycobacterium tuberculosis</i> Isolates in Moscow, Russia

<div><p>Background</p><p>The goal of this study was to compare the consistency of three assays for the determination of the drug resistance of <i>Mycobacterium tuberculosis</i> (MTB) strains with various resistance profiles isolated from the Moscow region.</p><p>Methods</p><p>A total of 144 MTB clinical isolates with a strong bias toward drug resistance were examined using Bactec MGIT 960, Sensititre MycoTB, and a microarray-based molecular assay TB-TEST to detect substitutions in the <i>rpoB</i>, <i>katG</i>, <i>inhA</i>, <i>ahpC</i>, <i>gyrA</i>, <i>gyrB</i>, <i>rrs</i>, <i>eis</i>, and <i>embB</i> genes that are associated with resistance to rifampin, isoniazid, fluoroquinolones, second-line injectable drugs and ethambutol.</p><p>Results</p><p>The average correlation for the identification of resistant and susceptible isolates using the three methods was approximately 94%. An association of mutations detected with variable resistance levels was shown. We propose a change in the breakpoint minimal inhibitory concentration for kanamycin to less than 5 μg/ml in the Sensititre MycoTB system. A pairwise comparison of the minimal inhibitory concentrations (MICs) of two different drugs revealed an increased correlation in the first-line drug group and a partial correlation in the second-line drug group, reflecting the history of the preferential simultaneous use of drugs from these groups. An increased correlation with the MICs was also observed for drugs sharing common resistance mechanisms.</p><p>Conclusions</p><p>The quantitative measures of phenotypic drug resistance produced by the Sensititre MycoTB and the timely detection of mutations using the TB-TEST assay provide guidance for clinicians for the choice of the appropriate drug regimen.</p></div

MICs based on the comparison of the MycoTB plate and MGIT 960 results.

<p>MICs based on the comparison of the MycoTB plate and MGIT 960 results.</p

MIC distributions of the clinical isolates characterized using the MGIT and TB-TEST assays.

<p>Resistant and susceptible isolates based on the MGIT results are indicated by the red and green lines, respectively. The light-red and light-green bars represent the numbers of resistant and susceptible isolates with mutations detected by the TB-TEST. The MGIT was not performed for rifabutin (RFB); therefore, only the distributions of all isolates and the isolates with mutations are shown.</p

Unusual Large-Scale Chromosomal Rearrangements in <i>Mycobacterium tuberculosis</i> Beijing B0/W148 Cluster Isolates

<div><p>The <i>Mycobacterium tuberculosis</i> (MTB) Beijing family isolates are geographically widespread, and there are examples of Beijing isolates that are hypervirulent and associated with drug resistance. One-fourth of Beijing genotype isolates found in Russia belong to the B0/W148 group. The aim of the present study was to investigate features of these endemic strains on a genomic level. Four Russian clinical isolates of this group were sequenced, and the data obtained was compared with published sequences of various MTB strain genomes, including genome of strain W-148 of the same B0/W148 group. The comparison of the W-148 and H37Rv genomes revealed two independent inversions of large segments of the chromosome. The same inversions were found in one of the studied strains after deep sequencing using both the fragment and mate-paired libraries. Additionally, inversions were confirmed by RFLP hybridization analysis. The discovered rearrangements were verified by PCR in all four newly sequenced strains in the study and in four additional strains of the same Beijing B0/W148 group. The other 32 MTB strains from different phylogenetic lineages were tested and revealed no inversions. We suggest that the initial largest inversion changed the orientation of the three megabase (Mb) segment of the chromosome, and the second one occurred in the previously inverted region and partly restored the orientation of the 2.1 Mb inner segment of the region. This is another remarkable example of genomic rearrangements in the MTB in addition to the recently published of large-scale duplications. The described cases suggest that large-scale genomic rearrangements in the currently circulating MTB isolates may occur more frequently than previously considered, and we hope that further studies will help to determine the exact mechanism of such events.</p></div

Genome rearrangements' representation for W-148 Progenitor I like H37Rv, W-148 Progenitor II and W-148 genomes.

<p>Each local collinear block (LCB) I–V is represented by a different color. Upside-down blocks (LCBs II and IV) represent the location of the reverse strand, which means an inversion has occurred. Asterisk indicates a terminus of a replication site. Terminus of a replication site was calculated based on GraphDNA (GC-skew mode) software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971-Thomas1" target="_blank">[19]</a>.</p

Schematic presentation of duplicated bases flanking the IS<i>6110</i> insertion sites between LCBs II&III and III&IV.

<p>Schematic presentation of duplicated bases flanking the IS<i>6110</i> insertion sites between LCBs II&III and III&IV.</p

Results of PCR verification of inversions.

<p>Electrophoregram of PCR products obtained for MTB strains during the amplification with primer sets 1–8 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone-0084971-t003" target="_blank">Table 3</a>).(A) SP 21 B0/W148 Beijing strain and (B) SP 5 non-B0/W148 Beijing strain. Lanes 1–8 correspond to primer sets 1–8; M is a marker GeneRuler 100 bp Plus DNA Ladder (Fermentas, SM0324); K- is a negative control.</p

Genotyping and drug resistance data of the B0/W148 strains sequenced in this study.

<p>¹ RIF - rifampicin, INH - isoniazid, EMB - ethambutol, STR - streptomycin, PZA - pyrazinamide, ETH - ethionamide, AMI- amikacin, CAPR - capreomycin, OFL – ofloxacin.</p><p>² B0 designation according to Narvskaya <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971-Narvskaya1" target="_blank">[6]</a>, W148 according to Bifani <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971-Bifani1" target="_blank">[14]</a>.</p><p>³ SITVITWEB was used for identification of data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971-Demay1" target="_blank">[15]</a>.</p><p>⁴ 24 – VNTR: s154, s580, s960, s1644, s2059, s2531, s2687, s2996, s3007, s3192, s4348, s802, s2165, s2461, s577, s2163, s4052, s4156, s424, s1955, s2347, s2401, s3171, s3690 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971-Supply1" target="_blank">[16]</a>.</p